Displaying alphavirus physicochemical consensus antigens on immunogenic liposomes enhances antibody elicitation in mice.

Cobalt-porphyrin phospholipid displays recombinant protein antigens on liposome surfaces via antigen polyhistidine-tag (His-tag), and when combined with monophosphorylated lipid A and QS-21 yields the "CPQ" vaccine adjuvant system. In this proof of principle study, CPQ was used to generate vaccine prototypes that elicited antibodies for two different alphaviruses (AV). Mice were immunized with computationally designed, His-tagged, physicochemical property consensus (PCPcon) protein antigens representing the variable B-domain of the envelope protein 2 (E2) from the serotype specific Venezuelan Equine Encephalitis Virus (VEEVcon) or a broad-spectrum AV-antigen termed EVCcon. The CPQ adjuvant enhanced the antigenicity of both proteins without eliciting detectable anti-His-tag antibodies. Antibodies elicited from mice immunized with antigens admixed with CPQ showed orders-of-magnitude higher levels of antigen-specific IgG compared to alternative control adjuvants. The ELISA results correlated with antiviral activity against VEEV strain TC83 and more weakly to Chikungunya virus 118/25. Thus, display of E.coli-produced His-tagged E2 protein segments on the surface of immunogenic liposomes elicits high levels of antigen-specific and AV neutralizing antibodies in mice with vaccination, while facilitating vaccine preparation and providing dose-sparing potential.

Liposomal Fba and Met6 peptide vaccination protects mice from disseminated candidiasis.

Epitopes from the Candida cell surface proteins Fba and Met6 are putative vaccine targets for invasive candidiasis. Here, we describe a Candida vaccine approach in which short peptides derived from Fba and Met6 are used in spontaneous nanoliposome antigen particle (SNAP) format. SNAP was enabled by the interaction of cobalt porphyrin phospholipid in liposomes with three histidine residues on the N-terminus of synthetic short peptide immunogens from Fba (F-SNAP), Met6 (M-SNAP), or bivalent Fba and Met6 (FM-SNAP). Liposomes were adjuvanted with synthetic monophosphoryl lipid and QS-21. In mice, immunization with F-SNAP, M-SNAP, or FM-SNAP induced antigen-specific IgG responses and mixed Th1/Th2 immunity. The duplex FM-SNAP vaccine elicited stronger antibody responses against each peptide, even at order-of-magnitude lower peptide dosing than a comparable adjuvanted, conjugate vaccine. Enzyme-linked immunosorbent spot analysis revealed the induction of antigen-specific, cytokine-producing T cells. Compared to F-SNAP or M-SNAP, higher production of TNFα, IL-2, and IFNγ was observed with re-stimulation of splenocytes from bivalent FM-SNAP-immunized mice. When vaccinated BALB/c mice were challenged with Candida auris, analysis of the fungal burden in the kidneys showed that SNAP vaccination protected from disseminated candidiasis. In a lethal fungal exposure model in A/J mice, F-SNAP, M-SNAP, and FM-SNAP vaccination protected mice from candidiasis challenge. Together, these results show that further investigation into the SNAP adjuvant platform is warranted using Fba and Met6 epitopes for a pan-Candida peptide vaccine that provides multifaceted protective immune responses. IMPORTANCE: This study introduces a promising vaccine strategy against invasive candidiasis, a severe fungal infection, by targeting specific peptides on the surface of Candida. Using a novel approach called spontaneous nanoliposome antigen particle (SNAP), we combined peptides from two key Candida proteins, Fba and Met6, into a vaccine. This vaccine induced robust immune responses in mice, including the production of protective antibodies and the activation of immune cells. Importantly, mice vaccinated with SNAP were shielded from disseminated candidiasis in experiments. These findings highlight a potential avenue for developing a broad-spectrum vaccine against Candida infections, which could significantly improve outcomes for patients at risk of these often deadly fungal diseases.

CoPoP liposomes displaying stabilized clade C HIV-1 Env elicit tier 2 multiclade neutralization in rabbits.

One of the strategies towards an effective HIV-1 vaccine is to elicit broadly neutralizing antibody responses that target the high HIV-1 Env diversity. Here, we present an HIV-1 vaccine candidate that consists of cobalt porphyrin-phospholipid (CoPoP) liposomes decorated with repaired and stabilized clade C HIV-1 Env trimers in a prefusion conformation. These particles exhibit high HIV-1 Env trimer decoration, serum stability and bind broadly neutralizing antibodies. Three sequential immunizations of female rabbits with CoPoP liposomes displaying a different clade C HIV-1 gp140 trimer at each dosing generate high HIV-1 Env-specific antibody responses. Additionally, serum neutralization is detectable against 18 of 20 multiclade tier 2 HIV-1 strains. Furthermore, the peak antibody titers induced by CoPoP liposomes can be recalled by subsequent heterologous immunization with Ad26-encoded membrane-bound stabilized Env antigens. Hence, a CoPoP liposome-based HIV-1 vaccine that can generate cross-clade neutralizing antibody immunity could potentially be a component of an efficacious HIV-1 vaccine.

The predominant Quillaja Saponaria fraction, QS-18, is safe and effective when formulated in a liposomal murine cancer peptide vaccine.

Extracts of the Chilean soapbark tree, Quillaja Saponaria (QS) are the source of potent immune-stimulatory saponin compounds. This study compared the adjuvanticity and toxicity of QS-18 and QS-21, assessing the potential to substitute QS-18 in place of QS-21 for vaccine development. QS-18, the most abundant QS saponin fraction, has been largely overlooked due to safety concerns. We found that QS-18 spontaneously inserted into liposomes, thereby neutralizing hemolytic activity, and following administration did not induce local reactogenicity in a footpad swelling test in mice. With high-dose intramuscular administration, transient weight loss was minor, and QS-18 did not induce significantly more weight loss compared to a liposome vaccine adjuvant system lacking it. Two days after administration, no elevation of inflammatory cytokines was detected in murine serum. In a formulation including cobalt-porphyrin-phospholipid (CoPoP) for short peptide sequestration, QS-18 did not impact the formation of peptide nanoparticles. With immunization, QS-18 peptide particles induced higher levels of cancer neoepitope-specific and tumor-associated antigen-specific CD8+ T cells compared to QS-21 particles, without indication of greater toxicity based on mouse body weight. T cell receptor sequencing of antigen-specific CD8+ T cells showed that QS-18 induced significantly more T cell transcripts. In two murine cancer models, vaccination with QS-18 peptide particles induced a similar therapeutic effect as QS-21 particles, without indication of increased toxicity. Antigen-specific CD8+ T cells in the tumor microenvironment were found to express the exhaustion marker PD-1, pointing to the rationale for exploring combination therapy. Taken together, these data demonstrate that QS-18, when formulated in liposomes, can be a safe and effective adjuvant to induce tumor-inhibiting cellular responses in murine models with potential to facilitate or diminish costs of production for vaccine adjuvant systems. Further studies are warranted to assess liposomal QS-18 immunogic, reactogenic and toxicological profiles in mice and other animal species.

Adjuvanted nanoliposomes displaying six hemagglutinins and neuraminidases as an influenza virus vaccine.

Inclusion of defined quantities of the two major surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA), could benefit seasonal influenza vaccines. Recombinant HA and NA multimeric proteins derived from three influenza serotypes, H1N1, H3N2, and type B, are surface displayed on nanoliposomes co-loaded with immunostimulatory adjuvants, generating "hexaplex" particles that are used to immunize mice. Protective immune responses to hexaplex liposomes involve functional antibody elicitation against each included antigen, comparable to vaccination with monovalent antigen particles. When compared to contemporary recombinant or adjuvanted influenza virus vaccines, hexaplex liposomes perform favorably in many areas, including antibody production, T cell activation, protection from lethal virus challenge, and protection following passive sera transfer. Based on these results, hexaplex liposomes warrant further investigation as an adjuvanted recombinant influenza vaccine formulation.

SARS-CoV-2 Protein Nanoparticle Vaccines Formed In Situ From Lyophilized Lipids.

The receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) glycoprotein is an appealing immunogen, but associated vaccine approaches must overcome the hapten-like nature of the compact protein and adapt to emerging variants with evolving RBD sequences. Here, a vaccine manufacturing methodology is proposed comprising a sterile-filtered freeze-dried lipid cake formulation that can be reconstituted with liquid proteins to instantaneously form liposome-displayed protein nanoparticles. Mannitol is used as a bulking agent and a small amount of Tween-80 surfactant is required to achieve reconstituted submicron particles that do not precipitate prior to usage. The lipid particles include an E. coli-derived monophosphoryl lipid A (EcML) for immunogenicity, and cobalt porphyrin-phospholipid (CoPoP) for antigen display. Reconstitution of the lipid cake with aqueous protein results in rapid conversion of the RBD into intact liposome-bound format prior to injection. Protein particles can readily be formed with sequent-divergent RBD proteins derived from the ancestral or Omicron strains. Immunization of mice elicits antibodies that neutralize respective viral strains. When K18-hACE2 transgenic mice are immunized and challenged with ancestral SARS-CoV-2 or the Omicron BA.5 variant, both liquid liposomes displaying the RBD and rapid reconstituted particles protect mice from infection, as measured by the viral load in the lungs and nasal turbinates.

The impact of green taxation on green low-carbon development in Yangtze River Delta region of China-based on mediating effect model and spatial Durbin model.

Promoting green low-carbon development is an important task in the construction of ecological civilization of China. The leadership has proclaimed green low-carbon development as the top national priority for development. Green low-carbon economy has become the high point of global industrial competition. Green taxation, as a tool for environmental regulation, plays an important role in the construction of ecological civilization. Therefore, this paper investigates the impact of green taxation on green low-carbon development in the Yangtze River Delta (YRD) region of China, using data of 41 cities from 2011 to 2021 in the YRD region. We construct a green low-carbon development index with the combined weight CRITIC-entropy method, and employ a mediating effect model and a spatial Durbin model based on double fixed effects in this study, which intends to provide a theoretical basis for the establishment of a sound tax policy system that is conducive to the green low-carbon development. The results show that (1) there is an evident spatial dependence of green low-carbon development in the YRD region; (2) green taxation has a significant positive effect on green low-carbon development through rationalization of industrial structure and advance of industrial structure; and (3) the positive impact of green taxation on green low-carbon development in the region is characterized by spatial spillover and heterogeneity. Specifically, green taxation significantly promotes green low-carbon development in the core area, but such green taxation dividend has yet to be realized in the expansion area.

Antibody induction in mice by liposome-displayed recombinant enterotoxigenic Escherichia coli (ETEC) colonization antigens.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) strains cause infectious diarrhea and colonize host intestine epithelia via surface-expressed colonization factors. Colonization factor antigen I (CFA/I), a prevalent ETEC colonization factor, is a vaccine target since antibodies directed to this fimbria can block ETEC adherence and prevent diarrhea. METHODS: Two recombinant antigens derived from CFA/I were investigated with a vaccine adjuvant system that displays soluble antigens on the surface of immunogenic liposomes. The first antigen, CfaEB, is a chimeric fusion protein comprising the minor (CfaE) and major (CfaB) subunits of CFA/I. The second, CfaEad, is the adhesin domain of CfaE. RESULTS: Owing to their His-tag, recombinant CfaEB and CfaEad, spontaneously bound upon admixture with nanoliposomes containing cobalt-porphyrin phospholipid (CoPoP), as well as a synthetic monophosphoryl lipid A (PHAD) adjuvant. Intramuscular immunization of mice with sub-microgram doses CfaEB or CfaEad admixed with CoPoP/PHAD liposomes elicited serum IgG and intestinal IgA antibodies. The smaller CfaEad antigen benefitted more from liposome display. Serum and intestine antibodies from mice immunized with liposome-displayed CfaEB or CfaEad recognized native CFA/I fimbria as evidenced by immunofluorescence and hemagglutination inhibition assays using the CFA/I-expressing H10407 ETEC strain. CONCLUSION: These data show that colonization factor-derived recombinant ETEC antigens exhibit immunogenicity when delivered in immunogenic particle-based formulations.

Assessing the implementation effect of Shanghai's compulsory municipal solid waste classification policy.

Waste treatment is a problem faced by cities all over the world. In recent years, China, as a developing country, regards the municipal solid waste (MSW) classification as one of the important strategies to deal with the MSW problem. The previous MSW classification policies in China were all only advocacy in nature. It was not until January 2019 that the "Regulations on the Management of MSW in Shanghai" was officially promulgated as China's first compulsory MSW classification policy, marking the beginning of an era of compulsory MSW classification in China. How effective is the implementation of Shanghai's compulsory MSW classification policy 18 months after its implementation and can developing countries continuously and effectively implement compulsory MSW classification policies? These are important issues of concern to the government, academia, and the public. This paper establishes a three-stage DEA model to evaluate the implementation effect of the compulsory MSW classification policies in Shanghai during the period of February 2019 and July 2020. The study found that the average efficiency of the compulsory MSW classification policy in Shanghai reached 0.906 during the study period, indicating that the policy was executed reasonably well. However, there are only 5 months in 18 months that the policy was fully effective (reaching efficiency level 1), suggesting that there is still room for improvement. The main reason for not being able to achieve full effectiveness in some months is attributed to scale efficiency. At the same time, the general public budget revenue and expenditure of environmental variables have positive and negative impacts on the policy implementation effect in Shanghai. The research results can provide experience for China to comprehensively implement the compulsory MSW classification policy in the future and can also provide valuable case study information for cities in other developing countries to implement the compulsory MSW classification policy.

Interim analysis from a phase 2 randomized trial of EuCorVac-19: a recombinant protein SARS-CoV-2 RBD nanoliposome vaccine.

BACKGROUND: Numerous vaccine strategies are being advanced to control SARS-CoV-2, the cause of the COVID-19 pandemic. EuCorVac-19 (ECV19) is a recombinant protein nanoparticle vaccine that displays the SARS-CoV-2 receptor-binding domain (RBD) on immunogenic nanoliposomes. METHODS: Initial study of a phase 2 randomized, observer-blind, placebo-controlled trial to assess the immunogenicity, safety, and tolerance of ECV19 was carried out between July and October 2021. Two hundred twenty-nine participants were enrolled at 5 hospital sites in South Korea. Healthy adults aged 19-75 without prior known exposure to COVID-19 were vaccinated intramuscularly on day 0 and day 21. Of the participants who received two vaccine doses according to protocol, 100 received high-dose ECV19 (20 µg RBD), 96 received low-dose ECV19 (10 µg RBD), and 27 received placebo. Local and systemic adverse events were monitored. Serum was assessed on days 0, 21, and 42 for immunogenicity analysis by ELISA and neutralizing antibody response by focus reduction neutralization test (FRNT). RESULTS: Low-grade injection site tenderness and pain were observed in most participants. Solicited systemic adverse events were less frequent, and mostly involved low-grade fatigue/malaise, myalgia, and headache. No clinical laboratory abnormalities were observed. Adverse events did not increase with the second injection and no serious adverse events were solicited by ECV19. On day 42, Spike IgG geometric mean ELISA titers were 0.8, 211, and 590 Spike binding antibody units (BAU/mL) for placebo, low-dose and high-dose ECV19, respectively (p < 0.001 between groups). Neutralizing antibodies levels of the low-dose and high-dose ECV19 groups had FRNT50 geometric mean values of 129 and 316, respectively. Boosting responses and dose responses were observed. Antibodies against the RBD correlated with antibodies against the Spike and with virus neutralization. CONCLUSIONS: ECV19 was generally well-tolerated and induced antibodies in a dose-dependent manner that neutralized SARS-CoV-2. The unique liposome display approach of ECV19, which lacks any immunogenic protein components besides the antigen itself, coupled with the lack of increased adverse events during boosting suggest the vaccine platform may be amenable to multiple boosting regimes in the future. Taken together, these findings motivate further investigation of ECV19 in larger scale clinical testing that is underway. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov as # NCT04783311.

Respiratory Vaccination with Hemagglutinin Nanoliposomes Protects Mice from Homologous and Heterologous Strains of Influenza Virus.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

Immune checkpoint blockade enhances chemophototherapy in a syngeneic pancreatic tumor model.

Pancreatic cancer (PaCa) suffers from poor treatment options for locally advanced cases. Chemophototherapy (CPT) is an emerging anti-tumor modality, and porphyrin-phospholipid liposomes have been shown to be versatile drug carriers for CPT in preclinical rodent models. Here we show that in the syngeneic subcutaneous KPC PaCa tumor model, exhausted CD8+ T cells are localized in the tumor, and that CPT is enhanced in combination with immune checkpoint blockade (ICB). Addition of ICB using anti-programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibodies resulted in ablation of medium-sized, established KPC tumors (∼200 mm3) without recurrence for over 100 days. Mice rejected subsequent tumor re-challenge. Flow cytometry and tumor slice analysis following injection of a fluorescently labeled anti-PD-1 antibody showed that CPT improved antibody delivery to the tumor microenvironment. Treatment of large established tumors (∼400 mm3) using with CPT and ICB induced appreciable tumor regression and delay in regrowth. Taken together, these data demonstrate the utility of combining CPT with immunotherapies.

Intranasal Immunization with Liposome-Displayed Receptor-Binding Domain Induces Mucosal Immunity and Protection against SARS-CoV-2.

The global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to efforts in developing effective vaccine approaches. Currently, approved coronavirus disease 2019 (COVID-19) vaccines are administered through an intramuscular (I.M.) route. Here, we show that the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD), when displayed on immunogenic liposomes, can be intranasally (I.N.) administered, resulting in the production of antigen-specific IgA and antigen-specific cellular responses in the lungs. Following I.N. immunization, antigen-presenting cells of the lungs took up liposomes displaying the RBD. K18 human ACE2-transgenic mice that were immunized I.M or I.N with sub-microgram doses of RBD liposomes and that were then challenged with SARS-CoV-2 had a reduced viral load in the early course of infection, with I.M. achieving complete viral clearance. Nevertheless, both vaccine administration routes led to full protection against lethal viral infection, demonstrating the potential for the further exploration and optimization of I.N immunization with liposome-displayed antigen vaccines.

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses.

Malaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1-2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

Vaccine co-display of CSP and Pfs230 on liposomes targeting two Plasmodium falciparum differentiation stages.

A vaccine targeting multiple stages of the Plasmodium falciparum parasite life cycle is desirable. The sporozoite surface Circumsporozoite Protein (CSP) is the target of leading anti-infective P. falciparum pre-erythrocytic vaccines. Pfs230, a sexual-stage P. falciparum surface protein, is currently in trials as the basis for a transmission-blocking vaccine, which inhibits parasite development in the mosquito vector. Here, recombinant full-length CSP and a Pfs230 fragment (Pfs230D1+) are co-displayed on immunogenic liposomes to induce immunity against both infection and transmission. Liposomes contain cobalt-porphyrin phospholipid (CoPoP), monophosphoryl lipid A and QS-21, and rapidly bind His-tagged CSP and Pfs230D1+ upon admixture to form bivalent particles that maintain reactivity with conformational monoclonal antibodies. Use of multicolor fluorophore-labeled antigens reveals liposome binding upon admixture, stability in serum and enhanced uptake in murine macrophages in vitro. Bivalent liposomes induce humoral and cellular responses against both CSP and Pfs230D1+. Vaccine-induced antibodies reduce parasite numbers in mosquito midguts in a standard membrane feeding assay. Mice immunized with liposome-displayed antigens or that passively receive antibodies from immunized rabbits have reduced parasite liver burden following challenge with transgenic sporozoites expressing P. falciparum CSP.

Elucidating functional epitopes within the N-terminal region of malaria transmission blocking vaccine antigen Pfs230.

Pfs230 is a leading malaria transmission blocking vaccine (TBV) candidate. Comprising 3135 amino acids (aa), the large size of Pfs230 necessitates the use of sub-fragments as vaccine immunogens. Therefore, determination of which regions induce functional antibody responses is essential. We previously reported that of 27 sub-fragments spanning the entire molecule, only five induced functional antibodies. A "functional" antibody is defined herein as one that inhibits Plasmodium falciparum parasite development in mosquitoes in a standard membrane-feeding assay (SMFA). These five sub-fragments were found within the aa 443-1274 range, and all contained aa 543-730. Here, we further pinpoint the location of epitopes within Pfs230 that are recognized by functional antibodies using antibody depletion and enrichment techniques. Functional epitopes were not found within the aa 918-1274 region. Within aa 443-917, further analysis showed the existence of functional epitopes not only within the aa 543-730 region but also outside of it. Affinity-purified antibodies using a synthetic peptide matching aa 543-588 showed activity in the SMFA. Immunization with a synthetic peptide comprising this segment, formulated either as a carrier-protein conjugate vaccine or with a liposomal vaccine adjuvant system, induced antibodies in mice that were functional in the SMFA. These findings provide key insights for Pfs230-based vaccine design and establish the feasibility for the use of synthetic peptide antigens for a malaria TBV.

Advanced Materials for SARS-CoV-2 Vaccines.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has killed untold millions worldwide and has hurtled vaccines into the spotlight as a go-to approach to mitigate it. Advances in virology, genomics, structural biology, and vaccine technologies have enabled a rapid and unprecedented rollout of COVID-19 vaccines, although much of the developing world remains unvaccinated. Several new vaccine platforms have been developed or deployed against SARS-CoV-2, with most targeting the large viral Spike immunogen. Those that safely induce strong and durable antibody responses at low dosages are advantageous, as well are those that can be rapidly produced at a large scale. Virtually all COVID-19 vaccines and adjuvants possess nanoscale or microscale dimensions and represent diverse and unique biomaterials. Viral vector vaccine platforms, lipid nanoparticle mRNA vaccines and multimeric display technologies for subunit vaccines have received much attention. Nanoscale vaccine adjuvants have also been used in combination with other vaccines. To deal with the ongoing pandemic, and to be ready for potential future ones, advanced vaccine technologies will continue to be developed in the near future. Herein, the recent use of advanced materials used for developing COVID-19 vaccines is summarized.

Lyophilized, thermostable Spike or RBD immunogenic liposomes induce protective immunity against SARS-CoV-2 in mice.

The COVID-19 pandemic has spurred interest in potent and thermostable SARS-CoV-2 vaccines. Here, we assess low-dose immunization with lyophilized nanoparticles decorated with recombinant SARS-CoV-2 antigens. The SARS-CoV-2 Spike glycoprotein or its receptor-binding domain (RBD; mouse vaccine dose, 0.1 µg) was displayed on liposomes incorporating a particle-inducing lipid, cobalt porphyrin-phospholipid (dose, 0.4 µg), along with monophosphoryl lipid A (dose, 0.16 µg) and QS-21 (dose, 0.16 µg). Following optimization of lyophilization conditions, Spike or RBD-decorated liposomes were effectively reconstituted and maintained conformational capacity for binding human angiotensin-converting enzyme 2 (hACE2) for at least a week when stored at 60°C in lyophilized but not liquid format. Prime-boost intramuscular vaccination of hACE2-transgenic mice with the reconstituted vaccine formulations induced effective antibody responses that inhibited RBD binding to hACE2 and neutralized pseudotyped and live SARS-CoV-2. Two days following viral challenge, immunized transgenic mice cleared the virus and were fully protected from lethal disease.

Immunization with short peptide particles reveals a functional CD8+ T-cell neoepitope in a murine renal carcinoma model.

BACKGROUND: Induction of CD8+ T cells that recognize immunogenic, mutated protein fragments in the context of major histocompatibility class I (MHC-I) is a pressing challenge for cancer vaccine development. METHODS: Using the commonly used murine renal adenocarcinoma RENCA cancer model, MHC-I restricted neoepitopes are predicted following next-generation sequencing. Candidate neoepitopes are screened in mice using a potent cancer vaccine adjuvant system that converts short peptides into immunogenic nanoparticles. An identified functional neoepitope vaccine is then tested in various therapeutic experimental tumor settings. RESULTS: Conversion of 20 short MHC-I restricted neoepitope candidates into immunogenic nanoparticles results in antitumor responses with multivalent vaccination. Only a single neoepitope candidate, Nesprin-2 L4492R (Nes2LR), induced functional responses but still did so when included within 20-plex or 60-plex particles. Immunization with the short Nes2LR neoepitope with the immunogenic particle-inducing vaccine adjuvant prevented tumor growth at doses multiple orders of magnitude less than with other vaccine adjuvants, which were ineffective. Nes2LR vaccination inhibited or eradicated disease in subcutaneous, experimental lung metastasis and orthotopic tumor models, synergizing with immune checkpoint blockade. CONCLUSION: These findings establish the feasibility of using short, MHC-I-restricted neoepitopes for straightforward immunization with multivalent or validated neoepitopes to induce cytotoxic CD8+ T cells. Furthermore, the Nes2LR neoepitope could be useful for preclinical studies involving renal cell carcinoma immunotherapy.

Position-Scanning Peptide Libraries as Particle Immunogens for Improving CD8+ T-Cell Responses.

Short peptides reflecting major histocompatibility complex (MHC) class I (MHC-I) epitopes frequently lack sufficient immunogenicity to induce robust antigen (Ag)-specific CD8+ T cell responses. In the current work, it is demonstrated that position-scanning peptide libraries themselves can serve as improved immunogens, inducing Ag-specific CD8+ T cells with greater frequency and function than the wild-type epitope. The approach involves displaying the entire position-scanning library onto immunogenic nanoliposomes. Each library contains the MHC-I epitope with a single randomized position. When a recently identified MHC-I epitope in the glycoprotein gp70 envelope protein of murine leukemia virus (MuLV) is assessed, only one of the eight positional libraries tested, randomized at amino acid position 5 (Pos5), shows enhanced induction of Ag-specific CD8+ T cells. A second MHC-I epitope from gp70 is assessed in the same manner and shows, in contrast, multiple positional libraries (Pos1, Pos3, Pos5, and Pos8) as well as the library mixture give rise to enhanced CD8+ T cell responses. The library mixture Pos1-3-5-8 induces a more diverse epitope-specific T-cell repertoire with superior antitumor efficacy compared to an established single mutation mimotope (AH1-A5). These data show that positional peptide libraries can serve as immunogens for improving CD8+ T-cell responses against endogenously expressed MHC-I epitopes.